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- EMDB-23320: WalkerB mutant human mitochondrial LONP1 bound to endogenous substrate -

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Basic information

Entry
Database: EMDB / ID: EMD-23320
TitleWalkerB mutant human mitochondrial LONP1 bound to endogenous substrate
Map dataFinal EM map of substrate-bound, WalkerB mutant human mitochondrial LONP1
Sample
  • Complex: WalkerB mutant Human mitochondrial LONP1
    • Protein or peptide: WalkerB mutant Human mitochondrial LONP1
Function / homology
Function and homology information


oxidation-dependent protein catabolic process / PH domain binding / endopeptidase La / G-quadruplex DNA binding / response to aluminum ion / mitochondrial DNA metabolic process / mitochondrial genome maintenance / ATP-dependent peptidase activity / protein quality control for misfolded or incompletely synthesized proteins / mitochondrial nucleoid ...oxidation-dependent protein catabolic process / PH domain binding / endopeptidase La / G-quadruplex DNA binding / response to aluminum ion / mitochondrial DNA metabolic process / mitochondrial genome maintenance / ATP-dependent peptidase activity / protein quality control for misfolded or incompletely synthesized proteins / mitochondrial nucleoid / insulin receptor substrate binding / chaperone-mediated protein complex assembly / DNA polymerase binding / regulation of peptidyl-tyrosine phosphorylation / negative regulation of insulin receptor signaling pathway / mitochondrion organization / response to hormone / proteolysis involved in protein catabolic process / ADP binding / protein catabolic process / single-stranded DNA binding / cellular response to oxidative stress / sequence-specific DNA binding / single-stranded RNA binding / response to hypoxia / mitochondrial matrix / serine-type endopeptidase activity / ATP hydrolysis activity / mitochondrion / nucleoplasm / ATP binding / membrane / identical protein binding / cytosol
Similarity search - Function
Lon protease homologue, chloroplastic/mitochondrial / Lon protease, bacterial/eukaryotic-type / Peptidase S16, active site / ATP-dependent serine proteases, lon family, serine active site. / Lon proteolytic domain profile. / Peptidase S16, Lon proteolytic domain / Lon protease / Lon protease (S16) C-terminal proteolytic domain / Lon protease, N-terminal domain superfamily / Lon N-terminal domain profile. ...Lon protease homologue, chloroplastic/mitochondrial / Lon protease, bacterial/eukaryotic-type / Peptidase S16, active site / ATP-dependent serine proteases, lon family, serine active site. / Lon proteolytic domain profile. / Peptidase S16, Lon proteolytic domain / Lon protease / Lon protease (S16) C-terminal proteolytic domain / Lon protease, N-terminal domain superfamily / Lon N-terminal domain profile. / Lon protease, N-terminal domain / ATP-dependent protease La (LON) substrate-binding domain / Found in ATP-dependent protease La (LON) / PUA-like superfamily / ATPase family associated with various cellular activities (AAA) / ATPase, AAA-type, core / Ribosomal protein S5 domain 2-type fold, subgroup / Ribosomal protein S5 domain 2-type fold / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Lon protease homolog, mitochondrial
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.8 Å
AuthorsShin M / Watson ER / Song AS / Novick SR / Griffin P / Wiseman RL / Lander GC
Funding support United States, 4 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute on Aging (NIH/NIA)AG067594 United States
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)NS095892 United States
National Institutes of Health/National Institute on Aging (NIH/NIA)AG061697 United States
National Institutes of Health/Office of the DirectorS10OD021634 United States
CitationJournal: Nat Commun / Year: 2021
Title: Structures of the human LONP1 protease reveal regulatory steps involved in protease activation.
Authors: Mia Shin / Edmond R Watson / Albert S Song / Jeffrey T Mindrebo / Scott J Novick / Patrick R Griffin / R Luke Wiseman / Gabriel C Lander /
Abstract: The human mitochondrial AAA+ protein LONP1 is a critical quality control protease involved in regulating diverse aspects of mitochondrial biology including proteostasis, electron transport chain ...The human mitochondrial AAA+ protein LONP1 is a critical quality control protease involved in regulating diverse aspects of mitochondrial biology including proteostasis, electron transport chain activity, and mitochondrial transcription. As such, genetic or aging-associated imbalances in LONP1 activity are implicated in pathologic mitochondrial dysfunction associated with numerous human diseases. Despite this importance, the molecular basis for LONP1-dependent proteolytic activity remains poorly defined. Here, we solved cryo-electron microscopy structures of human LONP1 to reveal the underlying molecular mechanisms governing substrate proteolysis. We show that, like bacterial Lon, human LONP1 adopts both an open and closed spiral staircase orientation dictated by the presence of substrate and nucleotide. Unlike bacterial Lon, human LONP1 contains a second spiral staircase within its ATPase domain that engages substrate as it is translocated toward the proteolytic chamber. Intriguingly, and in contrast to its bacterial ortholog, substrate binding within the central ATPase channel of LONP1 alone is insufficient to induce the activated conformation of the protease domains. To successfully induce the active protease conformation in substrate-bound LONP1, substrate binding within the protease active site is necessary, which we demonstrate by adding bortezomib, a peptidomimetic active site inhibitor of LONP1. These results suggest LONP1 can decouple ATPase and protease activities depending on whether AAA+ or both AAA+ and protease domains bind substrate. Importantly, our structures provide a molecular framework to define the critical importance of LONP1 in regulating mitochondrial proteostasis in health and disease.
History
DepositionJan 20, 2021-
Header (metadata) releaseJan 27, 2021-
Map releaseJan 27, 2021-
UpdateAug 11, 2021-
Current statusAug 11, 2021Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.23
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.23
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_23320.png

Downloads & links

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Map

FileDownload / File: emd_23320.map.gz / Format: CCP4 / Size: 144.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationFinal EM map of substrate-bound, WalkerB mutant human mitochondrial LONP1
Voxel sizeX=Y=Z: 1.15 Å
Density
Contour LevelBy AUTHOR: 0.23 / Movie #1: 0.23
Minimum - Maximum-0.39190757 - 0.8674516
Average (Standard dev.)0.000110262605 (±0.029041281)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions336336336
Spacing336336336
CellA=B=C: 386.4 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.151.151.15
M x/y/z336336336
origin x/y/z0.0000.0000.000
length x/y/z386.400386.400386.400
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ400400400
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS336336336
D min/max/mean-0.3920.8670.000

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Supplemental data

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Half map: Half-map of substrate-bound, WalkerB mutant human mitochondrial LONP1

Fileemd_23320_half_map_1.map
AnnotationHalf-map of substrate-bound, WalkerB mutant human mitochondrial LONP1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half-map of substrate-bound, WalkerB mutant human mitochondrial LONP1

Fileemd_23320_half_map_2.map
AnnotationHalf-map of substrate-bound, WalkerB mutant human mitochondrial LONP1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : WalkerB mutant Human mitochondrial LONP1

EntireName: WalkerB mutant Human mitochondrial LONP1
Components
  • Complex: WalkerB mutant Human mitochondrial LONP1
    • Protein or peptide: WalkerB mutant Human mitochondrial LONP1

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Supramolecule #1: WalkerB mutant Human mitochondrial LONP1

SupramoleculeName: WalkerB mutant Human mitochondrial LONP1 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: Complexes consisting of homohexameric LONP1 protease with an E591A mutation from Homo sapiens bound to endogenous co-purified substrate
Source (natural)Organism: Homo sapiens (human) / Organelle: Mitochondria / Location in cell: Matrix
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria) / Recombinant strain: Rosetta 2(DE3)pLysS / Recombinant plasmid: pET20b
Molecular weightTheoretical: 462 KDa

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Macromolecule #1: WalkerB mutant Human mitochondrial LONP1

MacromoleculeName: WalkerB mutant Human mitochondrial LONP1 / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: AIEEKFRERL KELVVPKHVM DVVDEELSKL GLLDNHSSEF NVTRNYLDWL TSIPWGKYSN ENLDLARAQA VLEEDHYGME DVKKRILEFI AVSQLRGSTQ GKILCFYGPP GVGKTSIARS IARALNREYF RFSVGGMTDV AEIKGHRRTY VGALKKTKTE NPLILIDAVD ...String:
AIEEKFRERL KELVVPKHVM DVVDEELSKL GLLDNHSSEF NVTRNYLDWL TSIPWGKYSN ENLDLARAQA VLEEDHYGME DVKKRILEFI AVSQLRGSTQ GKILCFYGPP GVGKTSIARS IARALNREYF RFSVGGMTDV AEIKGHRRTY VGALKKTKTE NPLILIDAVD KIGRGYQGDP SSALLELLDP EQNANFYLDV PVDLSKVLFI CTANVTDTIP EPLRDRMEMI NVSGYVAQEK LAIAERYLVP QARALCGLDE SKAKLSSDVL TLLIKQYCRE SGVRNLQKQV EKVLRKSAYK IVSGEAESVE VTPENLQDFV GKPVFTVERM VTPPGVVMGL AWTAMGGSTL FVETSLRRPG DKDGSLEVTG QLGEVMKESA RIAYTFARAF LMQHAPANDY LVTSHIHLHV PEGATPKDGP SAGCTIVTAL LSLAMGRPVR QNLAMTGEVS LTGKILPVGG IKEKTIAAKR AGVTCIVLPA ENKKDFYDLA AFITEGLEVH FVEHYREIFD IAF

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration2.5 mg/mL
BufferpH: 8
Component:
ConcentrationNameFormula
50.0 mMTris Base
75.0 mMPotassium ChlorideKCl
10.0 mMMagnesium ChlorideMgCl2
1.0 mMTCEP
1.0 mMAdenosine Triphosphate
0.263 mMBortezomib

Details: Solutions were made fresh from concentrated stocks and filtered using a 0.1 um syringe filter to avoid microbial contamination. Samples were mixed on ice and incubated with fresh ATP for 5 ...Details: Solutions were made fresh from concentrated stocks and filtered using a 0.1 um syringe filter to avoid microbial contamination. Samples were mixed on ice and incubated with fresh ATP for 5 minutes prior to vitrification.
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: GOLD / Support film - topology: HOLEY / Support film - Film thickness: 50.0 nm / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Atmosphere: OTHER
Details: Grids were plasma cleaned prior to sample application for 7 seconds using a Solarus plasma cleaner (Gatan, Inc.) with a 75% nitrogen, 25% oxygen atmosphere at 15W.
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 277 K / Instrument: HOMEMADE PLUNGER
Details: 4 uL of sample was applied per grid and manually blotted for 4 seconds followed by immediately plunge-freezing in liquid ethane cooled by liquid nitrogen..
DetailsThis sample was monodisperse.

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Calibrated defocus max: 1.5 µm / Calibrated defocus min: 0.5 µm / Calibrated magnification: 43478 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 1.2 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 36000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
TemperatureMin: 80.0 K / Max: 90.0 K
Alignment procedureComa free - Residual tilt: 0.14 mrad
DetailsComa-free alignment procedure from Herzik & Wu, Nature Methods (2017). Preliminary grid screening was performed manually prior to data collection.
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3710 pixel / Digitization - Dimensions - Height: 3838 pixel / Digitization - Sampling interval: 5.0 µm / Digitization - Frames/image: 0-58 / Number grids imaged: 1 / Number real images: 2415 / Average exposure time: 11.8 sec. / Average electron dose: 50.0 e/Å2
Details: Images were collected in counting mode at 5 frames per second.
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 938590
Details: Difference-of-Gaussians particle selection in Appion processing workflow was used to select particles.
CTF correctionSoftware - Name: CTFFIND (ver. 4) / Software - details: CTF correction was done using CTFFIND4 / Details: CTF parameters were estimated with CTFFind4
Startup modelType of model: OTHER
Details: An ab initio reconstruction of Human mitochondrial LONP1 was used as an initial model.
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.0.1)
Software - details: cryoSPARC 3.0.1 was used to assign initial euler angles
Final 3D classificationNumber classes: 3 / Avg.num./class: 76500 / Software - Name: cryoSPARC (ver. 3.0.1)
Software - details: cryoSPARC 3.0.1 was used to perform final classification
Details: The final 3D classification had an somewhat uneven distribution owing to preferred specimen orientation due to differential interactions with the air-water interface
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.0.1)
Software - details: cryoSPARC 3.0.1 was used to assign final euler angles
Details: cryoSPARC 3.0.1 was used to assign final angles
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 4.8 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.0.1)
Software - details: cryoSPARC 3.0.1 was used to perform final reconstruction
Number images used: 128929
FSC plot (resolution estimation)

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